Moraxella catarrhalis outer membrane protein-106 polypeptide, gene sequence and uses thereof

ABSTRACT

The invention discloses the  Moraxella catarrhalis  outer membrane protein-106 (OMP106) polypeptide, polypeptides derived therefrom (OMP106-derived polypeptides), nucleotide sequences encoding said polypeptides, and antibodies that specifically bind the OMP106 polypeptide and/or OMP106-derived polypeptides. Also disclosed are immunogenic, prophylactic or therapeutic compositions, including vaccines, comprising OMP106 polypeptide and/or OMP106-derived polypeptides. The invention additionally discloses methods of inducing immune responses to  M. catarrhalis  and  M. catarrhalis  OMP106 polypeptides and OMP106-derived polypeptides in animals.

1. INTRODUCTION

The present invention generally relates to the outer membraneprotein-106 (OMP106) polypeptide of Moraxella catarrhalis. The inventionencompasses a purified OMP106 polypeptide and polypeptides derivedtherefrom (OMP106-derived polypeptides). The invention also encompassesantibodies, including cytotoxic antibodies, that specifically bind theOMP106 polypeptide and/or OMP106-derived polypeptides. The inventionfurther encompasses prophylactic or therapeutic compositions, includingvaccines, that comprise OMP106 polypeptide and/or OMP106-derivedpolypeptides. The invention additionally provides methods of inducingimmune responses to M. catarrhalis in mammals. The invention furtherprovides isolated nucleotide sequences encoding the OMP106 polypeptideand OMP106-derived polypeptides, vectors having said sequences, and hostcells containing said vectors.

2. BACKGROUND OF THE INVENTION

Moraxella catarrhalis, also known as Moraxella (Branhamella) catarrhalisor Branhamella catarrhalis and formerly known as Neisseria catarrhalisor Micrococcus catarrhalis, is a gram-negative bacterium frequentlyfound in the respiratory tract of humans. M. catarrhalis, originallythought to be a harmless commensal organism, is now recognized as animportant pathogen in upper and lower respiratory tract infections inanimals. In humans, M. catarrhalis causes serious lower respiratorytract infections in adults with chronic lung disease, systemicinfections in immunocompromised patients, and otitis media and sinusitisin infants and children. See Helminen et al., 1993, Infect. Immun.61:2003-2010; Catlin, B. W., 1990, Clin. Microbiol. Rev. 3:293-320; andreferences cited therein.

2.1. Outer Membrane Proteins and Protective Antibodies

The outer surface components of Moraxella catarrhalis have been studiedin attempts to understand the pathogenic process of M. catarrhalisinfections and to develop useful therapeutic treatments and prophylacticmeasures against such infections. The outer membrane proteins (OMPs) inparticular have received considerable attention as possible virulencefactors and as potential vaccine antigens. M. catarrhalis has about 10to 20 different OMPs with 6 to 8 of these, OMPs A to H, as thepredominate species (Murphy and Loeb, 1989, Microbial Pathogen.6:159-174). The molecular weights of OMPs A to H range from 97 to 20 kD,respectively. See Bartos and Murphy, 1988, J. Infect. Dis. 158:761-765;Helminen et al., 1993, Infect. Immun. 61:2003-2010; Murphy et al, 1993,Molecul. Microbiol. 10: 87-97; and Sarwar et al, 1992, Infect. Immun.60:804-809. Comparisons of protein profiles by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of outer membranepreparations from 50 M. catarrhalis strains show nearly homogeneouspatterns of OMPs A to H (Bartos and Murphy, 1988, J. Infect. Dis.158:761-765).

In addition to OMPs A to H, a high molecular weight OMP, designatedHMW-OMP, having an apparent mass of 350 to 720 kD by SDS-PAGE has alsobeen identified as another prominent surface component present in manystrains of M. catarrhalis. HWM-OMP upon formic acid denaturationproduces a single band of 120 to 140 kD and, thus, appears to be anoligomeric protein (Klingman and Murphy, 1994, Infect. Immun.62:1150-1155). HMW-OMP appears to be the same protein as that designatedUspA by Helminen et al., (1994, J. Infect. Dis. 170:867-872) and shownto be present in a number of M. catarrhalis strains.

In intact bacterium or bacterially-derived outer membrane vesicles,several of the above-identified OMPs present surface-exposed epitopesthat elicit the production of antibodies that bind the OMPs. Theseantigenic OMPs include OMP E and OMP G (Murphy and Bartos, 1989, Infect.Immun. 57:2938-2941); OMP C/D (Sarwar et al., 1992, Infect. Immun.60:804-809); CopB, an 80 kD OMP, (Helminen et al., 1993, Infect. Immun.61:2003-2010); and UspA (Helminen et al., 1994, J. Infect. Dis.170:867-872).

The therapeutic potential of antibodies to surfaced-exposed epitopes ofCopB and UspA has been evaluated in an animal model. The model involveddirect bolus inoculation of lungs of BALB/c VAF/Plus mice with acontrolled number of M. catarrhalis cells and subsequent examination ofthe rate of pulmonary clearance of the bacteria (Unhanand et al., 1992,J. Infect. Dis. 165:644-650). Different clinical isolates of the M.catarrhalis exhibited different rates of clearance that correlated withthe level of granulocyte recruitment into the infection site. Passiveimmunization with a monoclonal antibody directed to a surface-exposedepitope of either CopB or UspA increased the rate of pulmonary clearanceof M. catarrhalis (Helminen et al., 1993, Infect. Immun. 61:2003-2010;Helminen et al., 1994, J. Infect. Dis. 170:867-872).

2.2. Bacterial/Host Cell Adherence and Hemagglutination

The adherence of bacterial pathogens to a host cell surface promotescolonization and initiates pathogenesis. See, E. H. Beachey, 1981, J.Infect. Dis. 143:325-345. Gram-negative bacteria typically expresssurface lectins that bind to specific oligosaccharides of glycoproteinsand/or glycolipids on the host cell surface. Such lectins are oftenassociated with pili or fimbriae. Bacterial adherence can also occur bynon-specific binding resulting from hydrophobic and/or chargeinteraction with the host cell surface.

The mechanism of M. catarrhalis adherence to cells of the respiratorytract remains poorly understood. The organism adheres to cultured humanoropharyngeal epithelial cells (Mbaki et al., 1987, Tohuku J. Exp. Med.153:111-121). A study by Rikitomi et al. suggests that fimbriae may havea role in the adherence to such cells as fimbriae denaturation ortreatment with anti-fimbriae antibodies reduced adherence by fimbriatedstrains (Rikitomi et al., 1991, Scand. J. Infect. Dis. 23:559-567).Fimbriae mediated binding, however, cannot be the sole basis of thisadherence as the most highly adhering strain, among the severalexamined, was a non-fimbriated strain.

Hemagglutination reactions often replace the more complicated adherenceassays in classifying bacterial adhesins. However, Rikitomi et al. foundno correlation between human oropharyngeal epithelial cell adherence andhemagglutination by M. catarrhalis strains (Id.). That is three highlyadhering strains did not agglutinate human erythrocytes. Thus, differentbinding mechanisms are involved in human oropharyngeal epithelial celladherence and hemagglutination.

By contrast, a recent study by Kellens et al. suggests thathemagglutination by M. catarrhalis is correlated with host celladherence (Kellens et al., 1995, Infection 23:37-41). However, thisstudy employed an adherence assay based on bacterial binding to porcinetracheal sections. It is unclear whether porcine tracheal tissue can beconsidered homologous to human respiratory tract tissue with respect toadherence by pathogenic strains of M. catarrhalis.

Notwithstanding the problematic adherence assay, Kellens et al. examinedthe hemagglutination activities of eighty-some clinical isolates of M.catarrhalis (Kellens et al., 1995, Infection 23:37-41). Nearlythree-quarters of the examined strains agglutinated human, rabbit,guinea pig, dog or rat erythrocytes, while the remaining strains didnot. The agglutination activities for some of the hemagglutinatingstains were further characterized and shown to be calcium ion dependentand inhibited by trypsin digestion or high-temperature treatment oraddition of D-glucosamine or D-galactosamine.

A survey of hemagglutinating and non-hemagglutinating M. catarrhalisstrains by Tucker et al. has shown that all strains bind the glycolipidgangliotetraosylceramide but only hemagglutinating strains bind theglycolipid globotetraosylceramide (Tucker et al., 1994, Annual Meetingof Amer. Soc. Microbiol., Abstract No. D124). Moreover, M. catarrhalishemagglutination activity was shown to be inhibited by variousmonosaccharides that comprise the carbohydrate moiety ofglobotetraosylceramide. These observations led Tucker et al. to suggestthat M. catarrhalis hemagglutinates by binding toglobotetraosylceramides in the cell membranes of susceptibleerythrocytes, including those of human red blood cells. To date, noprior art has identified a molecule on the outer surface of M.catarrhalis that is responsible for either host cell adherence orhemagglutination.

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an indication thatsuch reference is available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention encompasses the OMP106 polypeptide of M.catarrhalis and OMP106-derived polypeptides and methods for making saidpolypeptides. The invention also encompasses antisera and antibodies,including cytotoxic antibodies, specific for the OMP106 polypeptideand/or OMP106-derived polypeptides. The invention further encompassesimmunogenic, prophylactic or therapeutic compositions, includingvaccines, comprising one or more of said polypeptides. The inventionadditionally encompasses nucleotide sequences encoding saidpolypeptides. The invention further encompasses immunogenic,prophylactic or therapeutic compositions, including vaccines, comprisingan attentuated or inactivated non-hemagglutinating M. catarrhaliscultivar.

The present invention has many utilities. For example, the OMP106polypeptide and OMP106-derived polypeptides may be used as ligands todetect antibodies elicited in response to M. catarrhalis infections(e.g., in diagnosing M. catarrhalis infections). The OMP106 polypeptideand OMP106-derived polypeptides may also be used as immunogens forinducing M. catarrhalis-specific antibodies. Such antibodies are usefulin immunoassays to detect M. catarrhalis in biological specimens. Thecytotoxic antibodies of the invention are useful in passiveimmunizations against M. catarrhalis infections. The OMP106 polypeptideand OMP106-derived polypeptides may further be used as activeingredients in vaccines against M. catarrhalis infections.

The invention is based on the surprising discovery that hemagglutinatingM. catarrhalis strains and cultivars have an outer membrane protein,OMP106 polypeptide, which is about 180 kD to about 230 kD in molecularweight, and that non-hemagglutinating M. catarrhalis strains andcultivars either do not have OMP106 polypeptide or haveinappropriately-modified OMP106 polypeptide which is inactive inhemagglutination and not silver-stainable. The invention is furtherbased on the discovery that polyclonal antiserum induced by OMP106polypeptide isolated from a hemagglutinating M. catarrhalis strain hascytotoxic activity against a different hemagglutinating M. catarrhalisstrain but not against a non-hemagglutinating M. catarrhalis strain.

3.1. Definitions and Abbreviations

-   -   anti-OMP106=anti-OMP106 polypeptide antibody or antiserum    -   ATCC=American Type Culture Collection    -   blebs=naturally occurring outer membrane vesicles of M.        catarrhalis    -   Gb₄=GalNAcβ1-3Galα1-4Galβ1-4Glc1-1Ceramide    -   HA=hemagglutinating    -   immuno-reactive=capable of provoking a cellular or humoral        immune response    -   kD=kilodaltons    -   M. catarrhalis=Mc; Moraxella catarrhalis; Moraxella        (Branhamella) catarrhalis; Branhamella catarrhalis; Neisseria        catarrhalis; or Micrococcus catarrhalis    -   NHA=non-hemagglutinating    -   OG=n-octyl g-D-glucopyranoside or octyl glucoside    -   OMP106=the outer membrane protein-106 polypeptide of Moraxella        catarrhalis, having a molecular weight of about 180 kD to 230 kD        by SDS-PAGE; extractable from blebs or intact cells of M.        catarrhalis by OG or sarkosyl detergent    -   OMP106-derived    -   polypeptide fragment of the OMP106 polypeptide; variant of        wild-type OMP106 polypeptide or fragment thereof, containing one        or more amino acid deletions, insertions or substitutions; or        chimeric protein comprising a heterologous polypeptide fused to        the C-terminal or N-terminal or internal segment of a whole or a        portion of the OMP106 polypeptide    -   OMP=outer membrane protein    -   OMPs=outer membrane proteins    -   PBS=phosphate buffered saline    -   PAG polyacrylamide gel    -   polypeptide=a peptide of any length, preferably one having ten        or more amino acid residues    -   SDS=sodium dodecylsulfate    -   SDS-PAGE=sodium dodecylsulfate polyacrylamide gel        electrophoresis

Nucleotide or nucleic acid sequences defined herein are represented byone-letter symbols for the bases as follows:

-   A (adenine)-   C (cytosine)-   G (guanine)-   T (thymine)-   U (uracil)-   M (A or C)-   R (A or G)-   W (A or T/U)-   S(C or G)-   Y (C or T/U)-   K (G or T/U)-   V (A or C or G; not T/U)-   H (A or C or T/U; not G)-   D (A or G or T/U; not C)-   B (C or G or T/U; not A)-   N (A or C or G or T/U) or (unknown)

Peptide and polypeptide sequences defined herein are represented byone-letter symbols for amino acid residues as follows:

-   A (alanine)-   R (arginine)-   N (asparagine)-   D (aspartic acid)-   C (cysteine)-   Q (glutamine)-   E (glutamic acid)-   G (glycine)-   H (histidine)-   I (isoleucine)-   L (leucine)-   K (lysine)-   M (methionine)-   F (phenylalanine)-   P (proline)-   S (serine)-   T (threonine)-   W (tryptophan)-   Y (tyrosine)-   V (valine)-   X (unknown)

The present invention may be more fully understood by reference to thefollowing detailed description of the invention, non-limiting examplesof specific embodiments of the invention and the appended figures.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Denaturing PAGE comparison of outer membrane protein profiles ofM. catarrhalis blebs or octyl glucoside (OG) extracts of whole M.catarrhalis cells. The numbers over the lanes refer to the ATCC straindesignations. A prestained SDS-PAGE standard (BioRad catalog #161-0305)was used as molecular weight markers. The standard consisted of thefollowing polypeptides with their approximate molecular weights noted inparenthesis: rabbit muscle phosphorylase B (106 kD); bovine serumalbumin (80 kD); hen egg white ovalbumin (49.5 kD); bovine carbonicanhydrase (32.5 kD); soybean trypsin inhibitor (27.5 kD); hen egg whitelysozyme (18.5 kD). The positions of the molecular weight markers in thegel are noted on the left side of the drawing by arrows with themolecular weights (kD) of some of the markers above the arrows.

FIG. 2: Results from overlaying thin layer chromatograms of glycolipidswith ¹²⁵I-labeled outer membrane blebs. In Panels A-C, Lane 1 containsglycolipid standards indicated on the left; Lane 2 contains asialo-GM₁;Lane 3 contains Gb₃, Gb₄, and Forssman antigen; and Lane 4 contains aFolch extraction of human erythrocytes. The chromatogram shown in PanelA is stained with orcinol, the chromatogram shown in Panel B isoverlayed with ¹²⁵I-labeled blebs of ATCC strain 8176 (anon-hemagglutinating strain), and the chromatogram shown in Panel C isoverlayed with ¹²⁵I-labeled blebs of ATCC strain 49143 (ahemagglutinating strain). Only the hemagglutinating strain bound to theGb₄ glycolipid band in the third and fourth lanes.

FIG. 3: Protein profiles by silver staining of octyl glucoside extractsof outer membrane proteins following digestion of the M. catarrhaliscells with the proteases indicated in the figure. The hemagglutinationactivity of the cells following the digestion is indicated below thefigure in the row labeled HA. The molecular weight markers used are asper FIG. 1.

FIG. 4: Comparison of protein profiles by silver staining of outermembrane proteins from various ATCC strains of M. catarrhalis. Thestrain designations are indicated above the lanes. The hemagglutinationactivity of the strains are indicated in the row labeled HA below thefigure. Note a protein having an apparent molecular weight greater thanthat of rabbit muscle phosphorylase B (106 kD) is common to thehemagglutinating strains, but is absent in the non-hemagglutinatingstrains. This polypeptide is designated OMP106. The molecular weightmarkers used are as per FIG. 1.

FIG. 5: Comparison of protein profiles by silver staining of outermembrane proteins from two M. catarrhalis ATCC 49143 cultivars: 49143(hemagglutinating cultivar) and 49143-NHA (non-hemagglutinatingcultivar). The hemagglutination activities of the cultivars areindicated below the figure in the row labeled HA. Note the absence ofthe OMP106 polypeptide band (indicated by <) in the non-hemagglutinatingcultivar. The molecular weight markers used are as per FIG. 1.

FIG. 6: Molecular weight estimation of OMP106 in a 6% denaturingpolyacrylamide gel using OG extracts of ATCC strain 49143 that wereincubated in sample buffer at either 25° C. or 100° C. prior toapplication to the gel. Proteins in the gel were visualized by reductivesilver staining. Note that the OMP106 polypeptide band (indicated by the<) is seen only in the sample incubated at 100° C. A broad rangeSDS-PAGE standard (BioRad catalog #161-0317) was used as molecularweight markers. The standard consisted of the following polypeptides(approximate molecular weights noted in parenthesis): rabbit skeletalmuscle myosin (200 kD); E. coli β-galactosidase (116 kD); rabbit musclephosphorylase B (97.4 kD); bovine serum albumin (66.2 kD). The positionsof the molecular weight markers in the gel are noted on the right sideof the figure by arrows with the molecular weights (kD) of the markersabove the arrows.

FIG. 7: Southern blot analysis of DraI and HindIII restrictionendonuclease digests of M. catarrhalis chromosomal DNA probed withMc5-72. DNA of M. catarrhalis strain 49143 was digested with DraI orHindIII. Southern analysis of the digested DNA was carried out usingMc5-72 (SEQ ID NO:4) as the probe. The high stringency wash was 2×SSC,1% SDS at 50° C. for about 20 to about 30 minutes. Lane 1 containsHindIII digest; the hybridizing band has an approximate size of 8.0 kB.Lane 2 contains DraI digest: the hybridizing band has an approximatesize of 4.2 kB.

FIGS. 8A and 8B: Western Blots of protein extracts of M. catarrhalis andrelated species using a rabbit antiserum to OMP106 as the probe (FIG.8A), compared to the reactivity of the serum prior to immunization ofthe rabbit with OMP106 (FIG. 8B). Samples in the lanes of FIGS. 8A and8B are as follows: Lane A, M. catarrhalis; Lane B, Moraxella ovis; LaneC, Moraxella lacunata; Lane D, Moraxella osloensis; Lane E, Moraxellabovis; Lane F, Neisseria meningitidis; Lane G, Neisseria gonorrhoeae.The molecular weight markers used are as per FIG. 1.

FIG. 9A. Western blot demonstrating that a rabbit antiserum to theOMP106 polypeptide from M. catarrhalis ATCC 49143 cross-reacts with apolypeptide of a similar molecular weight in a number of HA and NHAstrains of M. catarrhalis (the location of the OMP106 polypeptide isindicated by the arrow). The Western examined octyl glucoside extractsof various M. catarrhalis strains. The ATCC accession numbers of thestrains are indicated at the top of the lanes. The transfer and Westernblot procedures used were identical to those used to obtain the blotsshown in FIG. 8.

FIG. 9B. Western blot of the same extracts as those in FIG. 9A using thepre-immune serum corresponding to that used in FIG. 9A.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Hemagglutinating andNon-Hemagglutinating Cultivars

The invention provides an isolated or a substantially pure OMP106polypeptide of M. catarrhalis. The OMP106 polypeptide comprises thewhole or a subunit of a protein embedded in or located on the outersurface of the outer membrane of hemagglutinating (HA) strains and manynonhemagglutinating (NHA) strains and cultivars of M. catarrhalis.OMP106 contributes directly or indirectly to the hemagglutinationphenotype of the HA strains and cultivars. According to the invention,HA M. catarrhalis cells agglutinate human or rabbit erythrocytes in anystandard hemagglutination assay, such as the one taught bySoto-Hernandez et al. 1989, J. Clin. Microbiol. 27:903-908. Although notintending to be limited to any particular mechanism of action, it ispresently envisaged that M. catarrhalis agglutinates erythrocytes bybinding to the globotetrose (Gb₄) moiety of glycolipid and glycoproteinreceptors on the host cell surfaces and that the hemagglutinationactivity is mediated in part by appropriately modified OMP106polypeptide, which has the particular property of being susceptible tosilver staining. By contrast, unmodified or inappropriately modifiedOMP106 polypeptide is neither active in mediating hemagglutination norsilver-stainable. Moreover, OMP106 polypeptide is the only polypeptidehaving an apparent molecular weight of about 180 kD to about 230 kD inSDS-PAGE that is OG- or sarkosyl-extractable from HA or NHA M.catarrhalis blebs or intact cells.

The hemagglutination activity of HA M. catarrhalis cells is inhibited byglobotetrose (GalNAcβ1-3Galβ1-4Galβ1-4Glcβ1; Gb₄) and themonosaccharides that comprise Gb4, including N-acetyl-D-galactosamine,D-galactose and glucose, and derivatives thereof, such asmethyl-∞-galactose or methyl-β-galactose. The hemagglutination activityof HA M. catarrhalis cells is also inhibited by relatively higherconcentrations of a number of other sugars including but not limited toD-mannose, L-fucose, D-glucose, and N-acetyl-D-glucosamine.

The hemagglutination activity and the OMP106 polypeptide of intact HA M.catarrhalis cells are both reduced or destroyed by digestion of intactM. catarrhalis cells by various proteases including, but not limited to,TLCK (Nα-ptosyl-L-lysine chloro methyl ketone [also known as1-chloro-3-tosylamino-7-amino-L-2-heptanone])-treated chymotrypsin,proteinase K and TPCK (N-tosyl-L-phenylalanine chloromethylketone)-treated trypsin. Protease V8 digestion of intact HA M.catarrhalis cells, however, affects neither the hemagglutinationactivity nor the physical integrity of the OMP106 polypeptide of suchcells.

A non-hemagglutinating (NHA) cultivar may be derived from a HA M.catarrhalis strain or cultivar by serial passage in static liquidcultures (i.e., liquid cultures maintained at 35° C. without shaking).For example, a HA M. catarrhalis strain or cultivar is grown in MuellerHinton broth and every five days an inoculum is taken from the surfaceof the static culture to inoculate a subsequent static culture. Thepreferred inoculum is any floating mat of cells at the surface of theculture. Passaging in static cultures is maintained until a NHA cultivaris produced. A NHA cultivar of the invention may be used to produceprotective vaccines, such as whole cell vaccines, against M. catarrhalisinfections.

By contrast, the hemagglutinating phenotype of a HA M. catarrhalisstrain or cultivar can be maintained by passaging the strain or cultivarin shaking liquid cultures. In an embodiment, a HA M. catarrhalis strainor cultivar is grown in Mueller Hinton broth at 35 to 37° C. withshaking at about 200 RPM and passaged every 24 to 48 hours. Thehemagglutinating phenotype of a HA M. catarrhalis strain or cultivaralso can be maintained by passaging on solid media. For example, a HA M.catarrhalis strain or cultivar is grown on a plate containing blood agaror Mueller Hinton agar.

5.2. OMP106 Polypeptide

OMP106 polypeptide of the invention is the sole outer membrane proteinof a HA M. catarrhalis strain or cultivar that has an apparent molecularweight in SDS-PAGE of about 180 kD to about 230 kD, preferably about 190kD. According to the invention, an outer membrane protein of M.catarrhalis is a polypeptide that is present in M. catarrhalis blebs, orthat can be extracted from M. catarrhalis blebs or intact cells byn-octyl β-D-glucopyranoside (OG) or sarkosyl detergent in buffersolution at room temperature. See Murphy and Loeb, 1989, MicrobialPathogenesis 6:159-174, for a discussion of M. catarrhalis blebs, whichare naturally occurring vesicles consisting of the outer membrane of M.catarrhalis. NHA M. catarrhalis strains or cultivars either do not haveOMP106 polypeptide, or have OMP106 polypeptide in a form that bindsanti-OMP106 antibodies (see Section 5.5., infra) but does not react withsilver stain (i.e., using Silver Stain Plus of BioRad [Richmond,Calif.], or the procedure described by Gottlieb and Chauko, 1987, Anal.Biochem. 165:33). By contrast, OMP106 polypeptide from HA M. catarrhalisstrains or cultivars binds anti-OMP106 antibodies, and reacts withsilver stain.

OMP106 polypeptide may be identified in HA M. catarrhalis blebs orintact cells by its susceptibility to degradation by protease treatmentthat also abolishes or attenuates the hemagglutination activity of thesame HA strain (See Section 5.1. above for examples of proteases that door do not destroy hemagglutination activity of intact M. catarrhaliscells). In other words, digestion with a protease that destroys orreduces the hemagglutination activity of a HA strain or cultivar willalso change, in SDS-PAGE, the abundance or the location of OMP106polypeptide isolated from the strain or cultivar after such a digestionas compared to that isolated from the same strain or cultivar before thedigestion.

OMP106 polypeptide may also be identified as the polypeptide in OG orsarkosyl extract of M. catarrhalis blebs or intact cells that has anapparent molecular weight of greater than 106 kD as determined bydenaturing gel electrophoresis in 12% PAG with SDS, using formulationsas described in Harlow and Lane (Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Appendix I,1988). Heat treatment of the OG or sarkosyl extract at 100° C. for 5minutes can cause the OMP106 polypeptide to have an apparent molecularweight of about 180 kD to about 230 kD as determined by SDS-PAGE in 6%PAG without any reducing agents, using formulations as described inHarlow and Lane, id. In a particular embodiment, OMP106 polypeptide inthe heat-treated OG or sarkosyl extract of M. catarrhalis strain ATCC49143 has an apparent molecular weight of about 190 kD.

In particular embodiments, the OMP106 polypeptide is that prepared fromany of M. catarrhalis strains including, but not limited to, ATCC 49143,ATCC 25238, ATCC 25240, ATCC 43617, ATCC 43618, ATCC 43627 and ATCC43628. The preferred source of OMP106 polypeptide is a HA cultivar ofsuch strains. The more preferred source is a HA cultivar of ATCC 49143.

In a particular embodiment, OMP106 polypeptide comprises, preferably atthe amino-terminal, the amino acid sequenceIGISEADGGKGGANARGDKSIAIGDIAQALGSQSIAIGDNKIV (SEQ ID NO:1) or a sequencesubstantially homologous thereto. The OMP106 polypeptide mayadditionally comprise, carboxyl-distal to the above mentioned sequence,an octapeptide having the amino acid sequence GTVLGGKK (SEQ ID NO:2) ora sequence substantially homologous thereto. As used herein asubstantially homologous amino acid sequence is at least 80%, preferably100%, identical to the referenced amino acid sequence.

According to various aspects of the invention, the polypeptides of theinvention are characterized by their apparent molecular weights based onthe polypeptides' migration in SDS-PAGE relative to the migration ofknown molecular weight markers. While any molecular weight standardsknown in the art may be used with the SDS-PAGE, preferred molecularweight markers comprise at least rabbit skeletal muscle myosin, E. coliβ-galactosidase and rabbit muscle phosphorylase B. One skilled in theart will appreciate that the polypeptides of the invention may migratedifferently in different types of gel systems (e.g., different buffers;different concentration of gel, crosslinker or SDS). One skilled in theart will also appreciate that the polypeptides may have differentapparent molecular weights due to different molecular weight markersused with the SDS-PAGE. Hence, the molecular weight characterization ofthe polypeptides of the invention is intended to be directed to coverthe same polypeptides on any SDS-PAGE systems and with any molecularweight markers which might indicate sightly different apparent molecularweights for the polypeptides than those disclosed here.

5.3. OMP106-Derived Polypeptides

An OMP106-derived polypeptide of the invention may be a fragment of theOMP106 polypeptide. The intact OMP106 polypeptide may contain one ormore amino acid residues that are not necessary to its immunogenicity.It may be the case, for example, that only the amino acid residuesforming a particular epitope of the OMP106 polypeptide is necessary forimmunogenic activity. Unnecessary amino acid sequences can be removed bytechniques well-known in the art. For example, the unwanted amino acidsequences can be removed by limited proteolytic digestion using enzymessuch as trypsin, papain, or related proteolytic enzymes or by chemicalcleavage using agents such as cyanogen bromide and followed byfractionation of the digestion or cleavage products.

An OMP106-derived polypeptide of the invention may also be a modifiedOMP106 polypeptide or fragment thereof (i.e., an OMP106 polypeptide orfragment having one or more amino acid substitutions, insertions and/ordeletions of the wild-type OMP106 sequence). Such modifications mayenhance the immunogenicity of the resultant polypeptide product or haveno effect on such activity. Modification techniques that may be usedinclude those disclosed in U.S. Pat. No. 4,526,716.

An OMP106-derived polypeptide may further be a chimeric polypeptidecomprising one or more heterologous polypeptides fused to theamino-terminal or carboxyl-terminal or internal of a complete OMP106polypeptide or a portion of or a fragment thereof. Useful heterologouspolypeptides comprising such chimeric polypeptide include, but are notlimited to, a) pre- and/or pro-sequences that facilitate the transport,translocation and/or processing of the OMP106-derived polypeptide in ahost cell, b) affinity purification sequences, and c) any usefulimmunogenic sequences (e.g., sequences encoding one or more epitopes ofa surface-exposed protein of a microbial pathogen).

Preferably, the OMP106-derived polypeptides of the invention areimmunologically cross-reactive with the OMP106 polypeptide, thus beingcapable of eliciting in an animal an immune response to M. catarrhalis.More preferably, the OMP106-derived polypeptides of the inventioncomprise sequences forming one or more outer-surface epitopes of thenative OMP106 polypeptide of M. catarrhalis (i.e., the surface-exposedepitopes of OMP106 polypeptide as it exists in intact M. catarrhaliscells). Such preferred OMP106-derived polypeptides can be identified bytheir ability to specifically bind antibodies raised to intact M.catarrhalis cells (e.g., antibodies elicited by formaldehyde orglutaldehyde fixed M. catarrhalis cells; such antibodies are referred toherein as “anti-whole cell” antibodies). For example, polypeptides orpeptides from a limited or complete protease digestion of the OMP106polypeptide are fractionated using standard methods and tested for theirability to bind anti-whole cell antibodies. Reactive polypeptidescomprise preferred OMP106-derived polypeptides. They are isolated andtheir amino acid sequences determined by methods known in the art.

Also preferably, the OMP106-derived polypeptides of the inventioncomprise sequences that form one or more epitopes of native OMP106polypeptide that mediate hemagglutination by HA M. catarrhalis cells.Such preferred OMP106-derived polypeptides may be identified by theirability to interfere with hemagglutination by HA M. catarrhalis cells.For example, polypeptides from a limited or complete protease digestionor chemical cleavage of OMP106 polypeptide are fractionated usingstandard methods and tested for the ability to interfere inhemagglutination by M. catarrhalis cells. Once identified and isolatedthe amino acid sequences of such preferred OMP106-derived polypeptidesare determined using standard sequencing methods. The determinedsequence may be used to enable production of such polypeptides bysynthetic chemical and/or genetic-engineering means.

These preferred OMP106-derived polypeptides also can be identified byusing anti-whole cell antibodies to screen bacterial librariesexpressing random fragments of M. catarrhalis genomic DNA or clonednucleotide sequences encoding the OMP106 polypeptide. See, e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., ColdSpring Harbor Press, NY, Vol. 1, Chapter 12. The reactive clones areidentified and their inserts are isolated and sequenced to determine theamino acid sequences of such preferred OMP106-derived polypeptides.

5.4. Isolation and Purification of OMP106 Polypeptide and OMP106-DerivedPolypeptides

The invention provides isolated OMP106 polypeptides and OMP106-derivedpolypeptides. As used herein, the term “isolated” means that the productis significantly free of other biological materials with which it isnaturally associated. That is, for example, an isolated OMP106polypeptide composition is between about 70% and 94% pure OMP106polypeptide by weight. Preferably, the OMP106 polypeptides andOMP106-derived polypeptides of the invention are purified. As usedherein, the term “purified” means that the product is substantially freeof other biological material with which it is naturally associated. Thatis comprising a purified OMP106 polypeptide composition is at least 95%pure OMP106 polypeptide by weight, preferably at least 98% pure OMP106polypeptide by weight, and most preferably at least 99% pure OMP106polypeptide by weight.

The OMP106 polypeptide of the invention may be isolated from proteinextracts including whole cell extract, of any M. catarrhalis strain orcultivar. Preferably, the protein extract is an octyl glucoside orsarkosyl extract of outer membrane vesicles (i.e., blebs) or whole cellsof M. catarrhalis including, but not limited to, any of strains TCC49143, ATCC 25238, ATCC 25240, ATCC 43617, ATCC 43618, TCC 43627 andATCC 43628. The preferred source of such extracts is a HA cultivar ofsuch strains. The more referred source of such extracts is a HA cultivarof ATCC 49143. Another source of the OMP106 polypeptide is proteinpreparations from gene expression systems expressing cloned sequencesencoding OMP106 polypeptide or OMP106-derived polypeptides (see Section5.8., infra).

The OMP106 polypeptide can be isolated and purified from the sourcematerial using any biochemical technique and approach well known tothose skilled in the art. In one approach, M. catarrhalis outer membraneis obtained by standard techniques and outer membrane proteins aresolubilized using a solubilizing compound such as a detergent. Apreferred solubilizing solution is one containing about 1.25% octylglucopyranoside w/v (OG). Another preferred solubilizing solution is onecontaining about 1.25% sarkosyl. OMP106 polypeptide is in thesolubilized fraction. Cellular debris and insoluble material in theextract are separated and removed preferably by centrifuging. Thepolypeptides in the extract are concentrated, incubated inSDS-containing Laemmli gel sample buffer at 100° C. for 5 minutes andthen fractionated by electrophoresis in a 6% denaturing sodiumdodecylsulfate (SDS) polyacrylamide gel (PAG) without reducing agent.See Laemmli, 1970, Nature 227:680-685. The band or fraction identifiedas OMP106 polypeptide as described above (e.g., the silver-stainedpolypeptide band that is present in the OG or sarkosyl extract of a HAbut not that of a corresponding NHA cultivar or that of the HA cultivarafter digestion with a protease that abolishes hemagglutinationactivity) may then be isolated directly from the fraction or gel slicecontaining the OMP106 polypeptide. In a preferred embodiment, OMP106polypeptide has an apparent molecular weight of 190 kD as determined bycomparing its migration distance or rate in a denaturing SDS-PAGErelative to those of rabbit skeletal muscle myosin (200 kD) and E. coliβ-galactosidase (116 kD).

Another method of purifying OMP106 polypeptide is by affinitychromatography using anti-OMP106 antibodies, (see Section 5.5).Preferably, monoclonal anti-OMP106 antibodies are used. The antibodiesare covalently linked to agarose gels activated by cyanogen bromide orsuccinamide esters (Affi-Gel, BioRad, Inc.) or by other methods known tothose skilled in the art. The protein extract is loaded on the top ofthe gel as described above. The contact is for a period of time andunder standard reaction conditions sufficient for OMP106 polypeptide tobind to the antibody. Preferably, the solid support is a material usedin a chromatographic column. OMP106 polypeptide is then removed from theantibody, thereby permitting the recovery OMP106 polypeptide inisolated, or preferably, purified form.

An OMP106-derived polypeptide of the invention can be produced bychemical and/or enzymatic cleavage or degradation of isolated orpurified OMP106 polypeptide. An OMP106-derived polypeptide can also bechemically synthesized based on the known amino acid sequence of OMP106polypeptide and, in the case of a chimeric polypeptide, those of theheterologous polypeptide by methods well-known in the art. See, forexample, Creighton, 1983, Proteins: Structures and Molecular Principles,W.H. Freeman and Co., NY.

An OMP106-derived polypeptide can also be produced in a gene expressionsystem expressing a recombinant nucleotide construct comprisingsequences encoding OMP106-derived polypeptides. The nucleotide sequencesencoding polypeptides of the invention may be synthesized, and/orcloned, and expressed according to techniques well known to thoseskilled in the art. See, for example, Sambrook, et al., 1989, MolecularCloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, NY,Chapter 9.

OMP106-derived polypeptides of the invention can be fractionated andpurified by the application of standard protein purification techniques,modified and applied in accordance with the discoveries and teachingsdescribed herein. In particular, preferred OMP106-polypeptides of theinvention, those that form an outer-surface epitope of the native OMP106polypeptide may be isolated and purified according to the affinityprocedures disclosed above for the isolation and purification of OMP106polypeptide (e.g., affinity purification using anti-OMP106 antibodies.

If desirable, the polypeptides of the invention may be further purifiedusing standard protein or peptide purification techniques including butare not limited to electrophoresis, centrifugation, gel filtration,precipitation, dialysis, chromatography (including ion exchangechromatography, affinity chromatography, immunoadsorbent affinitychromatography, reverse-phase high performance liquid chromatography,and gel permeation high performance liquid chromatography), isoelectricfocusing, and variations and combinations thereof.

One or more of these techniques may be employed sequentially in aprocedure designed to separate molecules according to their physical orchemical characteristics. These characteristics include thehydrophobicity, charge, binding capability, and molecular weight of theprotein. The various fractions of materials obtained after eachtechnique are tested for their abilities to bind the OMP106 receptor orligand, to bind anti-OMP106 antibodies or to interfere withhemagglutination by HA M. catarrhalis cells (“test” activities). Thosefractions showing such activity are then subjected to the next techniquein the sequential procedure, and the new fractions are tested again. Theprocess is repeated until only one fraction having the above described“test” activities remains and that fraction produces only a single bandor entity when subjected to polyacrylamide gel electrophoresis orchromatography.

5.5. OMP106 Immunogens and Anti-OMP106 Antibodies

The present invention provides antibodies that specifically bind OMP106polypeptide or OMP106-derived polypeptides. For the production of suchantibodies, isolated or preferably, purified preparations of OMP106polypeptide or OMP106-derived polypeptides are used as immunogens.

In an embodiment, the OMP106 polypeptide is separated from other outermembrane proteins present in the OG or sarksyl extract of outer membraneof HA M. catarrhalis cells or blebs using SDS-PAGE (see Section 5.2.above) and the gel slice containing OMP106 polypeptide is used as theimmunogen and injected into a rabbit to produce antisera containingpolyclonal OMP106 antibodies. The same immunogen can be used to immunizemice for the production of hybridoma lines that produce monoclonalanti-OMP106 antibodies. In particular embodiments, a PAG slicecontaining isolated or purified OMP106 from any of strains ATCC 49143,ATCC 25238, ATCC 25240, ATCC 43617, ATCC 43618, ATCC 43627 and ATCC43628 is used as the immunogen. In preferred embodiments, a PAG slicecontaining isolated or purified OMP106 from a HA cultivar of suchstrains is used. In a more preferred embodiment, a PAG slice containingisolated or purified OMP106 from a HA cultivar of strain ATCC 49143 isused as the immunogen.

In other embodiments, peptide fragments of OMP106 polypeptide are usedas immunogens. Preferably, peptide fragments of purified OMP106polypeptide are used. The peptides may be produced by proteasedigestion, chemical cleavage of isolated or purified OMP106 polypeptideor chemical synthesis and then may be isolated or purified. Suchisolated or purified peptides can be used directly as immunogens. Inparticular embodiments, useful peptide fragments include but are notlimited to those having the sequenceIGISEADGGKGGANARGDKSIAIGDIAQALGSQSIAIGDNKIV (SEQ ID NO:1) or any portionthereof that is 6 or more amino acids in length. In an anotherembodiment, the peptide fragment has the sequence GTVLGGKK (SEQ IDNO:2).

Useful immunogens may also comprise such peptides or peptide fragmentsconjugated to a carrier molecule, preferably a carrier protein. Carrierproteins may be any commonly used in immunology, include, but are notlimited to, bovine serum albumin (BSA), chicken albumin, keyhole limpethemocyanin (KLH) and the like. For a discussion of hapten proteinconjugates, see, for example, Hartlow, et al., Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1988, or a standard immunology textbook such as Roitt, I. et al.,IMMUNOLOGY, C. V. Mosby Co., St. Louis, Mo. (1985) or Klein, J.,IMMUNOLOGY, Blackwell Scientific Publications, Inc., Cambridge, Mass.,(1990).

In yet another embodiment, for the production of antibodies thatspecifically bind one or more outer-surface epitopes of the nativeOMP106 polypeptide, intact HA M. catarrhalis cells or blebs preparedtherefrom are used as immunogen. The cells or blebs may be fixed withagents such as formaldehyde or glutaldehyde before immunization. SeeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1988, Chapter 15. It ispreferred that such anti-whole cell antibodies be monoclonal antibodies.Hybridoma lines producing the desired monoclonal antibodies can beidentified by using purified OMP106 polypeptide as the screening ligand.Cells or blebs of any M. catarrhalis strain including, but not limitedto, ATCC 49143, ATCC 25238, ATCC 25240, ATCC 43617, ATCC 43618, ATCC43627 and ATCC 43628 are used as the immunogen for inducing theseantibodies. Preferably, cells or blebs of a HA cultivar of such strainsare used as the immunogen. More preferably, cells or blebs of a HAcultivar of strain ATCC 49143 are used as the immunogen for inducingthese antibodies.

Polyclonal antibodies produced by whole cell or bleb immunizationscontain antibodies that bind other M. catarrhalis outer membraneproteins (“non-anti-OMP106 antibodies”) and thus are more cumbersome touse where it is known or suspected that the sample contains other M.catarrhalis outer membrane proteins or materials that are cross-reactivewith these other outer membrane proteins. Under such circumstances, anybinding by the anti-whole cell antibodies of a given sample or band mustbe verified by coincidental binding of the same sample or band byantibodies that specifically bind OMP106 polypeptide (e.g., anti-OMP106)and/or a OMP106-derived polypeptide, or by competition tests usinganti-OMP106 antibodies, OMP106 polypeptide or OMP106-derived polypeptideas the competitor (i.e., addition of anti-OMP106 antibodies, OMP106polypeptide or OMP106-derived polypeptide to the reaction mix lowers orabolishes sample binding by anti-whole cell antibodies). Alternatively,such polyclonal antisera, containing “non-anti-OMP106” antibodies, maybe cleared of such antibodies by standard approaches and methods. Forexample, the non-anti-OMP106 antibodies may be removed by precipitationwith cells of NHA M. catarrhalis cultivars or M. catarrhalis strainsknown not to have the OMP106 polypeptide (e.g., ATCC 8176, morepreferably a NHA cultivar of ATCC 49143); or by absorption to columnscomprising such cells or outer membrane proteins of such cells.

In further embodiments, useful immunogens for eliciting antibodies ofthe invention comprise mixtures of two or more of any of theabove-mentioned individual immunogens.

Immunization of mammals with the immunogens described herein, preferablyhumans, rabbits, rats, mice, sheep, goats, cows or horses, is performedfollowing procedures well known to those skilled in the art, forpurposes of obtaining antisera containing polyclonal antibodies orhybridoma lines secreting monoclonal antibodies.

Monoclonal antibodies can be prepared by standard techniques, given theteachings contained herein. Such techniques are disclosed, for example,in U.S. Pat. No. 4,271,145 and U.S. Pat. No. 4,196,265. Briefly, ananimal is immunized with the immunogen. Hybridomas are prepared byfusing spleen cells from the immunized animal with myeloma cells. Thefusion products are screened for those producing antibodies that bind tothe immunogen. The positive hybridomas clones are isolated, and themonoclonal antibodies are recovered from those clones.

Immunization regimens for production of both polyclonal and monoclonalantibodies are well-known in the art. The immunogen may be injected byany of a number of routes, including subcutaneous, intravenous,intraperitoneal, intradermal, intramuscular, mucosal, or a combinationof these. The immunogen may be injected in soluble form, aggregate form,attached to a physical carrier, or mixed with an adjuvant, using methodsand materials well-known in the art. The antisera and antibodies may bepurified using column chromatography methods well known to those ofskill in the art.

According to the present invention, OMP106 polypeptides of M.catarrhalis strains, HA or NHA, are immuno-cross reactive. Thus,antibodies raised to OMP106 polypeptide of one M. catarrhalis strain orcultivar specifically bind OMP106 polypeptide and OMP106-derivedpolypeptides of other M. catarrhalis strains and cultivars. For example,polyclonal anti-OMP106 antibodies induced by OMP106 polypeptide ofstrain ATCC 49143 specifically bind not only the homologous OMP106polypeptide (i.e., the OMP106 polypeptide of strain ATCC 49143) but alsoOMP106 polypeptide and/or OMP106-derived polypeptides of other M.catarrhalis strains including, but not limited to, ATCC 43628, ATCC43627, ATCC 43618, ATCC 43617, ATCC 25240 and ATCC 25238.

The antibodies of the invention, including but not limited toanti-OMP106 antibodies, can be used to facilitate isolation andpurification of OMP106 polypeptide and OMP106-derived polypeptides. Theantibodies may also be used as probes for identifying clones inexpression libraries that have inserts encoding OMP106 polypeptide orfragments thereof. The antibodies may also be used in immunoassays(e.g., ELISA, RIA, Westerns) to specifically detect and/or quantitate M.catarrhalis in biological specimens. Anti-OMP106 antibodies of theinvention specifically bind OMP106 polypeptide and do not bind proteinsfrom related bacterial pathogens such as Moraxella ovis, Moraxellalacunata, Moraxella osloensis, Moraxella bovis, Neisseria meningitidis,Neisseria gonorrhoeae. Thus anti-OMP106 antibodies can be used todiagnose M. catarrhalis infections.

The antibodies of the invention, particularly those which are cytotoxic,may also be used in passive immunization to prevent or attenuate M.catarrhalis infections of animals, including humans. (As used herein, acytotoxic antibody is one which enhances opsinization and/or complementkilling of the bacterium bound by the antibody) An effectiveconcentration of polyclonal or monoclonal antibodies raised against theimmunogens of the invention may be administered to a host to achievesuch effects. The exact concentration of the antibodies administeredwill vary according to each specific antibody preparation, but may bedetermined using standard techniques well known to those of ordinaryskill in the art. Administration of the antibodies may be accomplishedusing a variety of techniques, including, but not limited to thosedescribed in Section 5.6. for the delivery of vaccines.

Prophylactic and therapeutic efficacies of the antibodies of theinvention can be determined by standard pharmaceutical procedures inexperimental animals. The data obtained from animal studies can be usedin formulating a range of dosages for use in humans.

5.6. Vaccines

The present invention also provides therapeutic and prophylacticvaccines against M. catarrhalis infections of animals, includingmammals, and more specifically rodents, primates, and humans. Thepreferred use of the vaccines is in humans. The vaccines can be preparedby techniques known to those skilled in the art and would comprise, forexample, the antigen in form of an immunogen, a pharmaceuticallyacceptable carrier, possibly an appropriate adjuvant, and possibly othermaterials traditionally found in vaccines. An immunologically effectiveamount of the immunogen to be used in the vaccine is determined by meansknown in the art in view of the teachings herein.

The vaccines of the present invention comprise an immunologicallyeffective amount of any of the immunogens disclosed in Section 5.5. in apharmaceutically acceptable carrier.

According to another embodiment, the vaccines of the invention comprisean immunologically effective amount of an inactivated or attenuated HAM. catarrhalis cultivar or NHA M. catarrhalis cultivar of the invention.An inactivated or attenuated HA M. catarrhalis cultivar or NHA M.catarrhalis cultivar is obtained using any methods known in the artincluding, but not limited to, chemical treatment (e.g., formalin), heattreatment and irradiation.

The term “immunologically effective amount” is used herein to mean anamount sufficient to induce an immune response which can prevent M.catarrhalis infections or attenuate the severity of any preexisting orsubsequent M. catarrhalis infections. The exact concentration willdepend upon the specific immunogen to be administered, but may bedetermined by using standard techniques well known to those skilled inthe art for assaying the development of an immune response.

Useful polypeptide immunogens include the isolated OMP106 polypeptideand OMP106-derived polypeptides. Preferred immunogens include thepurified OMP106 polypeptide and derived polypeptides or peptides ofOMP106. The combined immunogen and carrier may be an aqueous solution,emulsion or suspension. In general, the quantity of polypeptideimmunogen will be between 0.1 and 500 micrograms per dose. The carriersare known to those skilled in the art and include stabilizers, diluents,and buffers. Suitable stabilizers include carbohydrates, such assorbitol, lactose, manitol, starch, sucrose, dextran, and glucose andproteins, such as albumin or casein. Suitable diluents include saline,Hanks Balanced Salts, and Ringers solution. Suitable buffers include analkali metal phosphate, an alkali metal carbonate, or an alkaline earthmetal carbonate. The vaccine may also contain one or more adjuvants toimprove or enhance the immunological response. Suitable adjuvantsinclude, but are not limited to, peptides; aluminum hydroxide; aluminumphosphate; aluminum oxide; a composition that consists of a mineral oil,such as Marcol 52, or a vegetable oil and one or more emulsifyingagents, or surface active substances such as lysolecithin, polycations,polyanions; and potentially useful human adjuvants such as BCG andCorynebacterium parvum. The vaccine may also contain other immunogens.Such a cocktail vaccine has the advantage that immunity against severalpathogens can be obtained by a single administration. Examples of otherimmunogens are those used in the known DPT vaccines.

The vaccines of the invention are prepared by techniques known to thoseskilled in the art, given the teachings contained herein. Generally, animmunogen is mixed with the carrier to form a solution, suspension, oremulsion. One or more of the additives discussed above may be in thecarrier or may be added subsequently. The vaccine preparations may bedesiccated, for example, by freeze drying for storage purposes. If so,they may be subsequently reconstituted into liquid vaccines by theaddition of an appropriate liquid carrier.

The vaccines are administered to humans or other mammals, includingrodents and primates. They can be administered in one or more doses. Thevaccines may be administered by known routes of administration. Manymethods may be used to introduce the vaccine formulations describedhere. These methods include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, andintranasal routes. The preferred routes are intramuscular orsubcutaneous injection.

The invention also provides a method for inducing an immune response toM. catarrhalis in a mammal in order to protect the mammal againstinfection and/or attenuate disease caused by M. catarrhalis. The methodcomprises administering an immunologically effective amount of theimmunogens of the invention to the host and, preferably, administeringthe vaccines of the invention to the host.

5.7. Nucleic Acids Encoding OMP106 Polypeptide and OMP106-DerivedPolypeptides

The present invention also provides nucleic acids, DNA and RNA, encodingOMP106 polypeptide and OMP106-derived polypeptides. In one aspect, thenucleic acids of the invention may be synthesized using methods known inthe art. Specifically, a portion of or the entire amino acid sequence ofOMP106 polypeptide or an OMP106-derived polypeptide may be determinedusing techniques well known to those of skill in the art, such as viathe Edman degradation technique (see, e.g., Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., pp.34-49). The amino acid sequence obtained is used as a guide for thesynthesis of DNA encoding OMP106 polypeptide or OMP106-derivedpolypeptide using conventional chemical approaches or polymerase chainreaction (PCR) amplification of overlapping oligonucleotides.

In another aspect, the amino acid sequence may be used as a guide forsynthesis of oligonucleotide mixtures which in turn can be used toscreen for OMP106 polypeptide coding sequences in M. catarrhalis genomiclibraries. Such libraries may be prepared by isolating DNA from cells ofany M. catarrhalis strain. Preferably the DNA used as the source of theOMP106 polypeptide coding sequence, for both genomic libraries and PCRamplification, is prepared from cells of any M. catarrhalis strainincluding, but not limited to, ATCC 49143, ATCC 25238, ATCC 25240, ATCC43617, ATCC 43618, ATCC 43627 and ATCC 43628.

In the preparation of genomic libraries, DNA fragments are generated,some of which will encode parts or the whole of M. catarrhalis OMP106polypeptide. The DNA may be cleaved at specific sites using variousrestriction enzymes. Alternatively, one may use DNase in the presence ofmanganese to fragment the DNA, or the DNA can be physically sheared, asfor example, by sonication. The DNA fragments can then be separatedaccording to size by standard techniques, including but not limited to,agarose and polyacrylamide gel electrophoresis, column chromatographyand sucrose gradient centrifugation. The DNA fragments can then beinserted into suitable vectors, including but not limited to plasmids,cosmids, bacteriophages lambda or T₄, and yeast artificial chromosome(YAC). (See, for example, Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A PracticalApproach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) The genomic librarymay be screened by nucleic acid hybridization to labeled probe (Bentonand Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc.Natl. Acad. Sci. U.S.A. 72:3961).

The genomic libraries may be screened with a labeled degenerateoligonucleotide corresponding to the amino acid sequence of any peptideof OMP106 polypeptide using optimal approaches well known in the art. Inparticular embodiments, the screening probe is a degenerateoligonucleotide that corresponds to the peptide having the sequenceIGISEADGGKGGANARGDKSIAIGDIAQALGSQSIAIGDNKIV (SEQ ID NO:1) or a portionthereof. In another embodiment the screening probe may be a degenerateoligonucleotide that corresponds to a peptide having the sequenceGTVLGGKK (SEQ ID NO:2). In an additional embodiment, theoligonucleotides GGNACNGTNCTNGGNGGNAARAAR (SEQ ID NO:3) andGGNACNGTNTTRGGNGGNAARAAR (SEQ ID NO:7), each corresponding to OMP106peptide GTVLGGKK (SEQ ID NO:2), is used as the probe. In furtherembodiments, the sequenceGAAGCGGACGGGGGGAAAGGCGGAGCCAATGCGCGCGGTGATAAATCCATTGCTATTGGTGACATTGCGCAA (SEQ ID NO:4) or any fragments thereof, or any complement ofthe sequence or fragments may be used as the probe. Any probe usedpreferably is 15 nucleotides or longer.

Clones in libraries with insert DNA encoding the OMP106 polypeptide orfragments thereof will hybridize to one or more of the degenerateoligonucleotide probes. Hybridization of such oligonucleotide probes togenomic libraries are carried out using methods known in the art. Forexample, hybridization with the two above-mentioned oligonucleotideprobes may be carried out in 2×SSC, 1.0% SDS at 50° C. and washed usingthe same conditions. In a particular embodiment, ATCC 49143 DNA sequenceencoding the whole or a part of the OMP106 polypeptide is a HindIIIrestriction fragment of about 8,000 bp in length or a DRAI restrictionfragment of about 4,200 bp in length.

In yet another aspect, clones of nucleotide sequences encoding a part orthe entire OMP106 polypeptide or OMP106-derived polypeptides may also beobtained by screening M. catarrhalis expression libraries. For example,M. catarrhalis DNA is isolated and random fragments are prepared andligated into an expression vector (e.g., a bacteriophage, plasmid,phagemid or cosmid) such that the inserted sequence in the vector iscapable of being expressed by the host cell into which the vector isthen introduced. Various screening assays can then be used to select forthe expressed OMP106 polypeptide or OMP106-derived polypeptides. In oneembodiment, the various anti-OMP106 antibodies of the invention (seeSection 5.5) can be used to identify the desired clones using methodsknown in the art. See, for example, Harlow and Lane, 1988, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., Appendix IV. Clones or plaques from the library arebrought into contact with the antibodies to identify those clones thatbind.

In an embodiment, colonies or plaques containing DNA that encodes OMP106polypeptide or OMP106-derived polypeptide could be detected using DYNABeads according to Olsvick et al., 29th ICAAC, Houston, Tex. 1989,incorporated herein by reference. Anti-OMP106 antibodies are crosslinkedto tosylated DYNA Beads M280, and these antibody-containing beads wouldthen be used to adsorb to colonies or plaques expressing OMP106polypeptide or OMP106-derived polypeptide. Colonies or plaquesexpressing OMP106 polypeptide or OMP106-derived polypeptide isidentified as any of those that bind the beads.

Alternatively, the anti-OMP106 antibodies can be nonspecificallyimmobilized to a suitable support, such as silica or Celite™ resin. Thismaterial would then be used to adsorb to bacterial colonies expressingOMP106 polypeptide or OMP106-derived polypeptide as described in thepreceding paragraph.

In another aspect, PCR amplification may be used to producesubstantially pure DNA encoding a part of or the whole of OMP106polypeptide from M. catarrhalis genomic DNA. Oligonucleotide primers,degenerate or otherwise, corresponding to known OMP106 polypeptidesequences can be used as primers. In particular embodiments, anoligonucleotide, degenerate or otherwise, encoding the peptideIGISEADGGKGGANARGDKSIAIGDIAQALGSQSIAIGDNKIV (SEQ ID NO:1) or any portionthereof may be used as the 5′ primer. For example, a 5′ primer may bethe nucleotide sequenceGAAGCGGACGGGGGGAAAGGCGGAGCCAATGCGCGCGGTGATAAATCCATTGCTATTGGTGACATTGCGCAA (SEQ ID NO:4) or any portion thereof. Nucleotide sequences,degenerate or otherwise, that are reverse complements of sequenceencoding GTVLGGKK (SEQ ID NO:2) may be used as the 3′ primer. Forexample, an oligonucleotide, degenerate or otherwise, that has thedegenerate nucleotide sequence YTTYTTNCCNCCNAGNACNGTNCC (SEQ ID NO:6) orYTTYTTNCCNCCYAANACNGTNCC (SEQ ID NO:8) may be used as the 3′ primer inconjunction with the various 5′ primer discussed above.

PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermalcycler and Taq polymerase (Gene Amp™). One can choose to synthesizeseveral different degenerate primers, for use in the PCR reactions. Itis also possible to vary the stringency of hybridization conditions usedin priming the PCR reactions, to allow for greater or lesser degrees ofnucleotide sequence similarity between the degenerate primers and thecorresponding sequences in M. catarrhalis DNA. After successfulamplification of a segment of the sequence encoding OMP106 polypeptide,that segment may be molecularly cloned and sequenced, and utilized as aprobe to isolate a complete genomic clone. This, in turn, will permitthe determination of the gene's complete nucleotide sequence, theanalysis of its expression, and the production of its protein productfor functional analysis, as described infra.

Once an OMP106 polypeptide coding sequence has been isolated from one M.catarrhalis strain or cultivar, it is possible to use the same approachto isolate OMP106 polypeptide coding sequences from other M. catarrhalisstrains and cultivars. It will be recognized by those skilled in the artthat the DNA or RNA sequence encoding OMP106 polypeptide (or fragmentsthereof) of the invention can be used to obtain other DNA or RNAsequences that hybridize with it under conditions of moderate to highstringency, using general techniques known in the art. Hybridizationwith an OMP106 sequence from one M. catarrhalis strain or cultivar underhigh stringency conditions will identify the corresponding sequence fromother strains and cultivars. High stringency conditions vary with probelength and base composition. The formula for determining such conditionsare well known in the art. See Sambrook et al., 1989, Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Press, NY, Chapter 11. As usedherein high stringency hybridization conditions as applied to probes ofgreater than 300 bases in length involve a final wash in 0.1×SSC/0.1%SDS at 68° C. for at least 1 hour (Ausubel, et al., Eds., 1989, CurrentProtocols in Molecular Biology, Vol. I, Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., New York, at page 2.10.3). Inparticular embodiments, the high stringency wash in hybridization usinga probe having the sequence of SEQ ID NO:4 or its complement is 2×SSC,1% SDS at 50° C. for about 20 to about 30 minutes.

One skilled in the art would be able to identify complete clones ofOMP106 polypeptide coding sequence using approaches well known in theart. The extent of OMP106 polypeptide coding sequence contained in anisolated clone may be ascertained by sequencing the cloned insert andcomparing the deduced size of the polypeptide encoded by the openreading frames (ORFs) with that of OMP106 polypeptide and/or bycomparing the deduced amino acid sequence with that of known amino acidsequence of purified OMP106 polypeptide. Where a partial clone of OMP106polypeptide coding sequence has been isolated, complete clones may beisolated by using the insert of the partial clone as hybridizationprobe. Alternatively, a complete OMP106 polypeptide coding sequence canbe reconstructed from overlapping partial clones by splicing theirinserts together.

Complete clones may be any that have ORFs with deduced amino acidsequence matching that of OMP106 polypeptide or, where the completeamino acid sequence of the latter is not available, that of a peptidefragment of OMP106 polypeptide and having a molecular weightcorresponding to that of OMP106 polypeptide. Further, complete clonesmay be identified by the ability of their inserts, when placed in anexpression vector, to produce a polypeptide that binds antibodiesspecific to the amino-terminal of OMP106 polypeptide and antibodiesspecific to the carboxyl-terminal of OMP106 polypeptide.

Nucleic acid sequences encoding OMP106-derived polypeptides may beproduced by methods well known in the art. In one aspect, sequencesencoding OMP106-derived polypeptides can be derived from OMP106polypeptide coding sequences by recombinant DNA methods in view of theteachings disclosed herein. For example, the coding sequence of OMP106polypeptide may be altered creating amino acid substitutions that willnot affect the immunogenicity of the OMP106 polypeptide or which mayimprove its immunogenicity. Various methods may be used, including butnot limited to oligonucleotide directed, site specific mutagenesis.These and other techniques known in the art may be used to create singleor multiple mutations, such as replacements, insertions, deletions, andtranspositions, as described in Botstein and Shortle, 1985, Science229:1193-1210.

Further, DNA of OMP106 polypeptide coding sequences may be truncated byrestriction enzyme or exonuclease digestions. Heterologous codingsequence may be added to OMP106 polypeptide coding sequence by ligationor PCR amplification. Moreover, DNA encoding the whole or a part of anOMP-derived polypeptide may be synthesized chemically or using PCRamplification based on the known or deduced amino acid sequence ofOMP106 polypeptide and any desired alterations to that sequence.

The identified and isolated DNA containing OMP106 polypeptide orOMP106-derived polypeptide coding sequence can be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pBR322 or pUC plasmid derivatives. The insertion into a cloningvector can, for example, be accomplished by ligating the DNA fragmentinto a cloning vector which has complementary cohesive termini. However,if the complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved DNA may be modified byhomopolymeric tailing. Recombinant molecules can be introduced into hostcells via transformation, transfection, infection, electroporation,etc., so that many copies of the gene sequence are generated.

In an alternative method, the desired DNA containing OMP106 polypeptideor OMP106-derived polypeptide coding sequence may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired sequence, for example, by sizefractionation, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that contain OMP106 polypeptide or OMP106-derivedpolypeptide coding sequence enables generation of multiple copies ofsuch coding sequence. Thus, the coding sequence may be obtained in largequantities by growing transformants, isolating the recombinant DNAmolecules from the transformants and, when necessary, retrieving theinserted coding sequence from the isolated recombinant DNA.

5.8. Recombinant Production of OMP106 Polypeptide and OMP106-DerivedPolypeptides

OMP106 polypeptide and OMP106-derived polypeptides of the invention maybe produced through genetic engineering techniques. In this case, theyare produced by an appropriate host cell that has been transformed byDNA that codes for the polypeptide. The nucleotide sequence encodingOMP106 polypeptide or OMP106-derived polypeptides of the invention canbe inserted into an appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted polypeptide-coding sequence. The nucleotide sequencesencoding OMP106 polypeptide or OMP106-derived polypeptides is insertedinto the vectors in a manner that they will be expressed underappropriate conditions (e.g., in proper orientation and correct readingframe and with appropriate expression sequences, including an RNApolymerase binding sequence and a ribosomal binding sequence).

A variety of host-vector systems may be utilized to express thepolypeptide-coding sequence. These include but are not limited tomammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors, orbacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.Preferably, the host cell is a bacterium, and most preferably thebacterium is E. coli, B. subtilis or Salmonella.

The expression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.In a specific embodiment, a chimeric protein comprising OMP106polypeptide or OMP106-derived polypeptide sequence and a pre and/or prosequence of the host cell is expressed. In other specific embodiments, achimeric protein comprising OMP106 polypeptide or OMP106-derivedpolypeptide sequence and an affinity purification peptide is expressed.In further specific embodiments, a chimeric protein comprising OMP106polypeptide or OMP106-derived polypeptide sequence and a usefulimmunogenic peptide or polypeptide is expressed. In preferredembodiments, OMP106-derived polypeptide expressed contains a sequenceforming either an outer-surface epitope or the receptor-binding domainof native OMP106 polypeptide.

Any method known in the art for inserting DNA fragments into a vectormay be used to construct expression vectors containing a chimeric geneconsisting of appropriate transcriptional/translational control signalsand the polypeptide coding sequences. These methods may include in vitrorecombinant DNA and synthetic techniques and in vivo recombinants(genetic recombination). Expression of a nucleic acid sequence encodingOMP106 polypeptide or OMP106-derived polypeptide may be regulated by asecond nucleic acid sequence so that the inserted sequence is expressedin a host transformed with the recombinant DNA molecule. For example,expression of the inserted sequence may be controlled by anypromoter/enhancer element known in the art. Promoters which may be usedto control expression of inserted sequences include, but are not limitedto the SV40 early promoter region (Bernoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42) for expression in animalcells; the promoters of β-lactamase (Villa-Kamaroff et al., 1978, Proc.Natl. Acad. Sci. U.S.A. 75:3727-3731), tac (DeBoer et al., 1983, Proc.Natl. Acad. Sci. U.S.A. 80:21-25), λP_(L), or trc for expression inbacterial cells (see also “Useful proteins from recombinant bacteria” inScientific American, 1980, 242:74-94); the nopaline synthetase promoterregion or the cauliflower mosaic virus 35S RNA promoter (Gardner et al.,1981, Nucl. Acids Res. 9:2871), and the promoter of the photosyntheticenzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984,Nature 310:115-120) for expression implant cells; promoter elements fromyeast or other fungi such as the Gal4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter.

Expression vectors containing OMP106 polypeptide or OMP106-derivedpolypeptide coding sequences can be identified by three generalapproaches: (a) nucleic acid hybridization, (b) presence or absence of“marker” gene functions, and (c) expression of inserted sequences. Inthe first approach, the presence of a foreign gene inserted in anexpression vector can be detected by nucleic acid hybridization usingprobes comprising sequences that are homologous to the inserted OMP106polypeptide or OMP106-derived polypeptide coding sequence. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of foreign genes in the vector. For example, ifthe OMP106 polypeptide or OMP106-derived polypeptide coding sequence isinserted within the marker gene sequence of the vector, recombinantscontaining the insert can be identified by the absence of the markergene function. In the third approach, recombinant expression vectors canbe identified by assaying the foreign gene product expressed by therecombinant. Such assays can be based, for example, on the physical orfunctional properties of OMP106 polypeptide or OMP106-derivedpolypeptide in in vitro assay systems, e.g., binding to an OMP106 ligandor receptor, or binding with anti-OMP106 antibodies of the invention, orthe ability of the host cell to hemagglutinate or the ability of thecell extract to interfere with hemagglutination by M. catarrhalis.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Asexplained above, the expression vectors which can be used include, butare not limited to, the following vectors or their derivatives: human oranimal viruses such as vaccinia virus or adenovirus; insect viruses suchas baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), andplasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered OMP106 polypeptide orOMP106-derived polypeptide may be controlled. Furthermore, differenthost cells have characteristic and specific mechanisms for thetranslational and post-translational processing and modification ofproteins. Appropriate cell lines or host systems can be chosen to ensurethe desired modification and processing of the foreign proteinexpressed.

5.9. Reagents

The polypeptides, peptides, antibodies and nucleic acids of theinvention are useful as reagents for clinical or medical diagnosis of M.catarrhalis infections and for scientific research on the properties ofpathogenicity, virulence, and infectivity of M. catarrhalis, as well ashost defense mechanisms. For example, DNA and RNA of the invention canbe used as probes to identify the presence of M. catarrhalis inbiological specimens by hybridization or PCR amplification. The DNA andRNA can also be used to identify other bacteria that might encode apolypeptide related to the M. catarrhalis OMP106.

The polypeptides and peptides of the invention may be used to preparepolyclonal and monoclonal antibodies that can be used to further purifycompositions containing the polypeptides of the invention by affinitychromatography. The polypeptides and peptides can also be used instandard immunoassays to screen for the presence of antibodies to M.catarrhalis in a sample.

It is to be understood that the application of the teachings of thepresent invention to a specific problem or environment will be withinthe capabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the products of the presentinvention and processes for their preparation and use appear in thefollowing example.

6. EXAMPLE Isolation and Characterization of the OMP106 Polypeptide andGene Encoding same from Strain ATCC 491-430R Other Strains 6.1. Materialand Methods 6.1.1. Hemagglutination Assay

Hemagglutination by M. catarrhalis was tested as described bySoto-Hernandez et al. (J. Clin. Microbiol. 27:903-908) except 5%,instead of 3%, v/v erythrocytes were used in a slide agglutinationassay. Initial hemagglutination assays were performed using 20 μg ofbacterial cells (wet weight). Since M. catarrhalis ATCC strain 49143grown on blood agar plates at 35° C. gave a strong hemagglutinationreaction, it was selected as the reference strain. Serially dilutingATCC strain 49143 in 1:2 dilutions resulted in decreasinghemagglutination reactions. Scores of ++++ to + were based on thehemagglutination observed by ATCC strain 49143 after serial 1:2dilutions so that a + reaction resulted using ¼ the number of cellsrequired to achieve a +++reaction.

6.1.2. Inhibition of Hemagglutination

M. catarrhalis ATCC 49143 cell suspension was serially diluted 1:2, andthe dilution that yielded a+hemagglutination reaction when 7 μl ofDulbecco's phosphate buffered saline and 7 μl of 5% (v/v) human O⁺erythrocytes was used to assay inhibition of hemagglutination by simplesugars and sugar derivatives. To determine if simple sugars or sugarderivatives could inhibit hemagglutination by M. catarrhalis, 7 μl of agiven sugar at 500 mM was mixed with 7 μl of M. catarrhalis cells andincubated for 5 minutes to allow the sugar to interact with the cells.Then 7 μl of 5% (v/v) human O⁺ erythrocytes were added and thehemagglutination was scored after 1 minute. Each sugar and sugarderivative was tested for the ability to inhibit hemagglutination. Thenthe stock of each sugar and sugar derivative was serially diluted 1:2,and these dilutions were assayed for their ability to inhibithemagglutination using the procedure described above. In this manner,the minimal concentration of carbohydrate required to inhibithemagglutination was determined.

6.1.3. Ligand and Receptor Binding

M. catarrhalis binding to animal cell glycolipid receptors was examinedusing thin layer chromatography (TLC) fractionation of the host cellglycolipids and labeled cell overlay of the chromatogram following theprocedures described by Magnani et al., 1982, J. Biol. Chem.257:14365-14369. Briefly, glycolipids obtained from Matreya Inc.(Pleasant Gap, Pa.) were resolved on high performance thin layerchromatograph plates (HPTLC) in chloroform, methanol, water (5:4:1) Theplates were either stained with orcinol at 100° C., or were overlaidwith ¹²⁵I-labeled M. catarrhalis blebs prepared as previously described(Murphy and Loeb, 1989, Microbial Pathogen. 6:159-174) at 2×10⁶ cpm/mlfor 2 hours. The plates were then washed 5 times, dried and exposed toX-ray film.

6.1.4. OG Extraction of OMPS

Strains of M. catarrhalis were each grown at 35° C. at 200 rpm in 1liter of Mueller Hinton broth in a 4 liter flask. Outer membrane protein(OMP) preparations were isolated by treating 50 mg of cells (wet weight)with 0.67 ml of 1.25% n-octyl β-D-glucopyranoside (i.e., octylglucoside; OG) in phosphate buffered saline (PBS) for 30 minutes at roomtemperature. Cells were pelleted in a microcentrifuge for 5 minutes andthe supernatant was used as an octyl glucoside extract. Comparison ofprotein profiles of these extracts from a number of strains of M.catarrhalis to those of blebs (i.e., outer membrane vesicles) isolatedby differential centrifugation, which are highly enriched for outermembrane proteins (OMPs) from M. catarrhalis (Murphy and Loeb, 1989,Microbial Pathogen. 6:159-174) indicates the octyl glucoside extractscontain predominately outer membrane proteins of M. catarrhalis (FIG.1). This indicated that octyl glycoside extraction provided a more rapidprocedure with a higher yield of outer membrane proteins as compared toouter membrane proteins prepared from blebs.

6.1.5. Proteolytic Digestion of OMP106

50 mg of cells from ATCC strain 49143 in 1 ml of Dulbecco's phosphatebuffered saline were digested for 1 hour at room temperature with thefollowing proteases: TLCK-treated chymotrypsin (5 mg), Proteinase K (5mg), TPCK-treated trypsin (5 mg), or protease V8 (100 Units). Allproteases were obtained from Sigma Chemicals (St. Louis, Mo.).Immediately following the protease treatment, cells were washed once inPBS and resuspended in 1 ml of PBS and the hemagglutinating activity wastested. Additionally, protease-treated bacterial cells were extractedwith octyl glucoside so the outer membrane proteins could be resolved toidentify specific proteins that may have been digested by the proteases.

6.1.6. Non-Hemagglutinating Cultivars

Normally, hemagglutinating M. catarrhalis cultures are grown in shakerflasks containing Mueller Hinton Broth at 35 to 37° C. at 200 rpm for 24to 48 hours. Cells taken directly from a blood agar plate or an agarplate of Mueller Hinton media also express the hemagglutinatingphenotype. To select for a non-hemagglutinating (NHA) cultivar, ATCCstrain 49143 was serially passaged every 5 days in static cultures grownin Mueller Hinton broth at 35° C. With each passage, inoculum was takenonly from the surface of the broth culture. By the second passage, afloating mat of cells had developed and this mat of cells was used asthe inoculum for subsequent cultures. Serial culturing in this mannerproduced NHA cultivars of ATCC 49143 typically after three passages.

6.1.7. Isolation of OMP106 Polypeptide

OMP106 polypeptide from outer membrane extract of M. catarrhalis ATCC49143 is detected (e.g., by silver staining or anti-OMP106 antibodies)in denaturing gels only after the extract has been incubated at 100° C.for five minutes. In order to determine if the appearance of the OMP106band after incubation at 100° C. is the result of lower molecular weightproteins aggregating during boiling, or if the boiling allows a normallyaggregated protein to enter the gel, an unboiled octyl glucoside outermembrane extract of ATCC 49143 was analyzed on a native polyacrylamidegel. Specific regions of the gel including that immediately below thesample well were excised and boiled. The resulting samples were thenresolved on a denaturing polyacrylamide gel and stained with silverstain (Silver Stain Plus, Catalog number 161-0449, BioRad Laboratories,Richmond, Calif.). For N-terminal sequencing, an octyl glucoside outermembrane extract of ATCC 49143 was mixed with PAGE sample buffercontaining SDS, and was incubated for 5 minutes in boiling water bath.The proteins were then resolved on a 12% PAG with SDS and transferred toa PVDF membrane by electroblotting. The region of the membranecontaining the OMP106 band was then cut out for amino-terminalsequencing. None of the PAGE procedures used to isolate the OMP106polypeptide used reducing agents in the sample or gel buffers.

6.1.8. Anti-OMP106 Antiserum

Antiserum to OMP106 were prepared by resolving OMP106 polypeptide from aHA cultivar of ATCC 49143 in a denaturing sodium dodecylsulfatepolyacrylamide gel as previously described (Lammeli, 1970, Nature227:680-685), and cutting the OMP106-containing band out of the gel. Theexcised band was macerated and injected into a rabbit to generateantiserum to OMP106 polypeptide. The antiserum was used to inhibithemagglutination as described in section 6.1.2. supra, but using theantiserum in place of the carbohydrate. The antiserum was also examinedfor complement-mediated cytotoxic activity against M. catarrhalis asdescribed in section 7.

6.1.9. Western Blots with Anti-OMP106 Antiserum

M. catarrhalis ATCC 49143, ATCC 43628, ATCC 43627, ATCC 43618, ATCC43617, ATCC 25240, ATCC 25238, and ATCC 8176; M. ovis ATCC 33078; M.lacunata ATCC 17967; M. bovis ATCC 10900; M. osloensis ATCC 10973;Neisseria gonorrhoeae (clinical isolate); and N. meningitidis ATCC 13077were grown on chocolate agar plates for 48 hours at 35° C. in 5% CO₂.Cells were removed by scraping the colonies from the agar surface usinga polystyrene inoculating loop. Cells were then solubilized bysuspending 30 μg of cells in 150 μl of PAGE sample buffer (360 mM Trisbuffer [pH 8.8], containing 4% sodium dodecylsulfate and 20% glycerol),and incubating the suspension at 1001° C. for 5 minutes. The solubilizedcells were resolved on 12% polyacrylamide gels as per Laemmli and theseparated proteins were electrophoretically transferred to PVDFmembranes at 100 V for 1.5 hours as previously described (Thebaine etal. 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354) except 0.05% sodiumdodecylsulfate was added to the transfer buffer to facilitate themovement of proteins from the gel. The PVDF membranes were thenpretreated with 25 ml of Dulbecco's phosphate buffered saline containing0.5% sodium casein, 0.5% bovine serum albumin and 1% goat serum. Allsubsequent incubations were carried out using this pretreatment buffer.

PVDF membranes were incubated with 25 ml of a 1:500 dilution ofpreimmune rabbit serum or serum from a rabbit immunized with OMP106polypeptide (as described above) for 1 hour at room temperature. PVDFmembranes were then washed twice with wash buffer (20 mM Tris buffer [pH7.5.] containing 150 mM sodium chloride and 0.05% Tween-20). PVDFmembranes were incubated with 25 ml of a 1:5000 dilution ofperoxidase-labeled goat anti-rabbit IgG (Jackson ImmunoResearchLaboratories, West Grove Pa. Catalog number 111-035-003) for 30 minutesat room temperature. PVDF membranes were then washed 4 times with washbuffer, and were developed with 3,3′diaminobenzidine tetrahydrochlorideand urea peroxide as supplied by Sigma Chemical Co. (St. Louis, Mo.catalog number D-4418) for 4 minutes each.

6.1.10. Anti-OMP106 Immunofluorescence Staining of Cell Surface

M. catarrhalis ATCC 49143 was grown overnight at 35° C. in a shakingwater bath in Mueller Hinton broth. The cells were pelleted bycentrifugation and then resuspended in an equal volume of Dulbecco'smodification of phosphate buffered saline without calcium or magnesium(PBS/MC). 20 μl of the cell suspension was applied to each of 5 cleanmicroscope slides. After setting for 10 seconds, the excess fluid wasremoved with a micropipettor, and the slides were allowed to air dry for1 hour. The slides were then heat fixed over an open flame until theglass was warm to the touch. The slides were initially treated with 40μl of 1:40 dilution of anti-OMP106 antiserum or preimmune serum from thesame animal diluted in PBS/MC, or PBS/MC for 10 minutes, then washed 5times with PBS/MC. The slides were treated with 40 μl of 5 μg/ml PBS/MCof fluorescein isothiocyanate-labeled goat antibody to rabbit IgG(Kirkegaard and Perry Laboratories, Inc, Gaithersburg, Md. catalognumber 02-15-06). The slides were incubated in the dark for 10 minutesand were washed 5 times in PBS/MC. Each slide was stored covered withPBS/MC under a cover slide and was viewed with a fluorescence microscopefitted with a 489 nm filter. For each sample five fields-of-view werevisually examined to evaluate the extent of straining.

6.2. Results 6.2.1. Hemagglutination Activity

The agglutination activity of M. catarrhalis with respect toerythrocytes is species specific with the strongest activity observedwith human erythrocytes. Rabbit erythrocytes are also agglutinated by M.catarrhalis, but less dramatically than are human cells. Theerythrocytes from mouse, horse or sheep were not agglutinated (see Table1).

TABLE 1 Strength of hemagglutination of erythrocytes from variousspecies using M. catarrhalis ATCC 49143 Source of Score for erythrocyteshemagglutination^(a) Human ++++ Rabbit ++ Mouse − Horse − Sheep −^(a)++++ = strongest agglutination, − indicates no agglutination

6.2.2. OMP106 Receptors and Ligands

M. catarrhalis hemagglutination activity is due to binding toglobotetrose (Gb₄). Blebs from hemagglutinating strains bind to aglycolipid having Gb₄₁ whereas non-hemagglutinating strains do not bindto the same glycolipid (see FIG. 2). M. catarrhalis hemagglutinationactivity is inhibited by monosaccharide constituents of Gb₄ orderivatives of such monosaccharides, with the most potent inhibitorsbeing N-acetyl galactosamine and galactose (especially the alpha anomerof the galactose) (see Table 2).

TABLE 2 The minimum concentration of sugars required to inhibithemagglutination (MIC) by M. catarrhalis Sugar MIC (mM)* D-Glucose >167D-Mannose 83 D-Galactose 41 L-Fucose 83 N-acetyl-D-Glucosamine >167N-acetyl-D-Galactosamine 41 Methyl-α-Glucose >167 Methyl-α-Mannose 167Methyl-α-Galactose 10 Methyl-β-galactose 83 *Minimal concentration ofsugar required to inhibit a 1+ hemagglutination reaction by M.catarrhalis ATCC 49143 with 5% washed human O+ erythrocytes.

Both N-acetyl galactosamine and alpha-galactose are part of the Gb₄tetrasaccharide. The correlation between hemagglutination and binding toGb₄, and the observation that hemagglutination is inhibited bymonosaccharides that comprise the Gb₄ receptor suggest that M.catarrhalis cells bind to the tetrasaccharide Gb₄. This tetrasaccharideis present on human erythrocytes and tissues, and could mediate M.catarrhalis attachment to eukaryotic membranes.

6.2.3. Identification of OMP106 Polypeptide

Proteolytic digestion of M. catarrhalis cells, and subsequent analysisof hemagglutination by the digested cells demonstrated that proteasetreatment with chymotrypsin and proteinase K destroyed thehemagglutination activity, and treatment with trypsin partiallydestroyed hemagglutination activity, indicating the hemagglutinatingactivity is protein mediated. Analysis of the OMP protein profiles ofprotease digested M. catarrhalis cells showed that multiple proteins hadbeen degraded in each sample, so the profiles did not provide a clue asto which protein is directly responsible for or indirectly contributedto the hemagglutination activity (see FIG. 3).

Since protease treatment indicated a polypeptide is directly orindirectly responsible for hemagglutination activity, we used SDS-PAGEto compare the OMP profiles from hemagglutinating strains with the OMPprofiles from non-hemagglutinating strains (FIG. 4). Analysis of thedifferences between these profiles indicated that the hemagglutinatingstrains had two unique polypeptides, one with an apparent molecularweight of 27 kD (designated OMP27) and the other was the only proteinwith an apparent molecular weight of greater than 106 kD (designatedOMP106). Notably, the OMP106 polypeptide band was absent in the OMPpreparations of various protease treated cells that have reduced or nohemagglutination activity, whereas the OMP27 band was present in the OMPpreparation of proteinase K treated cells that have no hemagglutinationactivity. Additionally, the OMP106 polypeptide band was not degraded byproteinase V8 digestion, which did not affect hemagglutination activityof treated cells.

6.2.4. OMP Profile of NHA Cultivars

Serial culturing of NHA cultivar of ATCC 49143 in static culture at 35°C. produced a NHA cultivar (designated 49143-NHA) by the third passageof the culture. This loss of the hemagglutination activity wasrepeatable. Analysis of OMP profiles of OG outer membrane extracts ofthe HA and NHA cultivars showed that the OMP106 polypeptide band wasmissing from the 49143-NHA extract (FIG. 5). This suggested that OMP106polypeptide is the M. catarrhalis hemagglutinin (i.e., OMP106polypeptide binds Gb₄ receptor or is a subunit of a homopolymericprotein that binds Gb₄ receptor) or forms a part of the M. catarrhalishemagglutinin (i.e., OMP106 polypeptide is a subunit of aheteropolymeric protein that binds Gb₄ receptor).

6.2.5. OMP106 and Hemagglutination

Polyclonal antiserum raised to ATCC 49143 OMP106 polypeptide neutralizedhemagglutination by ATCC 49143, as well as that by heterologous ATCC43627. This further supports the conclusion that M. catarrhalishemagglutinating activity comprises OMP106 polypeptide, and that OMP106polypeptide is antigenically conserved among strains. See also FIG. 9A,which shows antibodies in the polyclonal antiserum binding OMP106polypeptide of heterologous M. catarrhalis strains.

6.2.6. Outer Surface Location of OMP106

Rabbit anti-OMP106 antiserum was used in indirect immunofluorescencestaining to determine if OMP106 polypeptide is exposed on the outersurface of M. catarrhalis cells. M. catarrhalis cells treated withanti-OMP106 antiserum stained more intensely and uniformly than didcells treated with preimmune serum or PBS/MC. This indicated that inintact M. catarrhalis cells OMP106 polypeptide was reactive withanti-OMP106 antibodies. This result indicates that OMP106 polypeptide isexposed on the outer surface of M. catarrhalis. This finding isconsistent with OMP106 polypeptide having a role in hemagglutinationand, moreover, indicates that OMP106 polypeptide would be useful as avaccine.

6.2.7. Properties of OMP106 Polypeptide

OMP106 polypeptide exists as a large protein complex in its native stateor aggregates when extracted with octyl glucoside. This conclusion issupported by the finding that extracting M. catarrhalis cells with octylglucoside will solubilize OMP106 polypeptide, but the extracted OMP106polypeptide does not enter denaturing PAGs unless the extract is firstincubated at 100° C. (FIG. 6). Further, the OMP106 polypeptide band doesnot appear to form from lower molecular weight polypeptides thatpolymerize or aggregate upon heating, since OMP106 polypeptide in anon-heat denatured sample is trapped in the sample well and enters theresolving gel only if the sample has been first incubated at 100° C.This biochemical property is very useful for identifying OMP106polypeptide in various gels.

Using octyl glucoside extracts of M. catarrhalis, then incubating theextracts with sodium dodecyl sulfate at 100° C., and resolving theproteins on a denaturing polyacrylamide gel, we have estimated theapparent molecular weight of OMP106 polypeptide from various strains ofM. catarrhalis, specifically those of ATCC 25238, ATCC 25240, ATCC43617, ATCC 43618, ATCC 43627 and ATCC 43628, to range from about 180 kDto about 230 kD (FIG. 9A), whereas the OMP106 polypeptide of strain ATCC49143 appears to have an apparent weight of about 190 kD (FIG. 6).

OMP106 polypeptide of strain ATCC 49143 was extracted from the gel sliceand its N-terminal was sequenced. The sequencing showed the N-terminalof OMP106 polypeptide from the outer membrane of ATCC 49143 to beIGISEADGGKGGANARGDKSIAIGDIAQALGSQSIAIGDNKIV (SEQ ID NO:1). Additionally,an internal peptide of OMP106 produced by Lys-C digest (Fernandez etal., 1994, Anal Biochem 218:112-117) has been isolated and its sequencedetermined to be GTVLGGKK (SEQ ID NO:2).

We generated three oligonucleotide probes. Two probes correspond to theinternal peptide GTVLGGKK, one has the following sequenceGGNACNGTNCTNGGNGGNAARAAR (SEQ ID NO:3), the other has the followingsequence GGNACNGTNTTRGGNGGNAARAAR (SEQ ID NO:7). The other probe, Mc5-72, encoding an internal fragment (SEQ ID NO:5) of the amino-terminalsequence of OMP106 (SEQ ID NO:1) has the following sequenceGAAGCGGACGGGGGGAAAGGCGGAGCCAATGCGCGCGGTGATAAATCCATTGCTATTGGTGACATTGCGCAA (SEQ ID NO:4). Hybridization of the Mc 5-72 probe to acomplete HindIII or DraI digest of M. catarrhalis DNA in each instanceproduced a single band in Southern blot analysis (FIG. 7). Thehybridizing band in the HindIII digest has an approximate size of 8.0kb; the hybridizing band in the DraI digest has an approximate size of4.2 kb (FIG. 7).

6.2.8. Conservation of OMP106 Polypeptide

Western blot analysis of outer membrane protein extracts of a number ofM. catarrhalis strains and related species of bacteria showed that theanti-OMP106 antibodies binds to a polypeptide of about 180 Kd to about230 kD in many M. catarrhalis strains, both HA and NHA strains orcultivars (FIG. 9A). The anti-OMP106 antibodies did not bind to anypolypeptide in the protein extracts of related bacteria (FIG. 8A). Theseresults demonstrate the following: 1) Anti-OMP106 antibodies may be usedto specifically identify and distinguish M. catarrhalis from relatedspecies of bacteria. 2) OMP106 polypeptide may be used to generateantibodies that have diagnostic application for identification of M.catarrhalis. 3) Antibodies to OMP106 polypeptide of one strain (e.g.,OMP106 of ATCC 49143) may be used to identify and isolate thecorresponding OMP106 polypeptide of other M. catarrhalis strains.Interestingly, the Western blot results show that many of the NHA M.catarrhalis strains have OMP106 polypeptide in OG extracts of theirouter membranes. This finding and the fact that silver staining of OMPsfrom OG outer membrane extracts of NHA M. catarrhalis strains after PAGEdoes not reveal a band in the 180 kD to 230 kD range indicate thatOMP106 polypeptide is expressed by most M. catarrhalis strains orcultivars but that, in order to be active in hemagglutination (i.e.,binding to receptor on mammalian cell surfaces) or silver stainable, theOMP106 polypeptide must be appropriately modified in some manner.Apparently only HA strains and cultivars are capable of appropriatelymodifying OMP106 polypeptide so that it can mediate bacterial binding tohemagglutinin receptor on mammalian cell surfaces.

7. EXAMPLE Efficacy of OMP106 Vaccine: Cytotoxic Activity of Anti-OMP106Antiserum

Complement-mediated cytotoxic activity of anti-OMP106 antibodies wasexamined to determine the vaccine potential of OMP106 polypeptide.Antiserum to OMP106 polypeptide of a HA cultivar of ATCC 49143 wasprepared as described in Section 6.1.8. supra. The activities of thepre-immune serum and the anti-OMP106 antiserum in mediating complementkilling of M. catarrhalis were examined using the “Serum BactericidalTest” described by Zollinger et al. (Immune Responses to Neiserriameningitis, in Manual of Clinical Laboratory Immunology, 3rd ed., pg347-349), except that cells of HA and NHA M. catarrhalis strains orcultivars were used instead of Neiserria meningitis cells.

The results show that anti-OMP106 antiserum mediated complement-killingof a HA cultivar of heterologous M. catarrhalis ATCC 43627 but not a NHAcultivar of M. catarrhalis ATCC 43627 or the NHA M. catarrhalis ATCC8176. Table 3 summarizes the complement mediated cytotoxic activities ofpre-immune serum and anti-OMP106 antiserum against a HA cultivar of ATCC43627.

TABLE 3 Complement mediated cytotoxic activities of pre- immune serumand anti-OMP106 antiserum Cytotoxic Titer¹ Pre-immune Anti-OMP106Experiment 1 16 128 Experiment 2 8 64 ¹The titer is in the highestdilution at which a serum can mediate complement killing of a HAcultivar of ATCC 43627 (e.g., 16 represents a 16 fold dilution of theserum), the larger the number, the higher the cytotoxic activity ortiter.

As shown in Table 3, the anti-OMP106 antiserum has 8 fold greatercytotoxic activity than the pre-immune serum. This finding indicatesthat OMP106 polypeptide is useful as a vaccine against HA M. catarrhalisstrains and cultivars.

Although the invention is described in detail with reference to specificembodiments thereof, it will be understood that variations which arefunctionally equivalent are within the scope of this invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-12. (canceled)
 13. An isolated antibody that specifically binds OMP106polypeptide, which is an outer membrane polypeptide of Moraxellacatarrhalis having a molecular weight of about 180 kD as determined inSDS polyacrylamide gel electrophoresis using rabbit skeletal musclemyosin and E. coli β-galactosidase as the 200 kD and 116.25 kD molecularweight standards, respectively.
 14. An isolated antibody thatspecifically binds OMP106 polypeptide or fragment thereof comprising asequence substantially homologous to the sequence of SEQ ID NO:1.
 15. Anisolated antibody according to claim 14 that specifically binds theOMP106 polypeptide or a fragment thereof comprising the sequence of SEQID NO:
 1. 16. The isolated antibody of claim 13, which is a cytotoxicantibody that mediates complement killing of Moraxella catarrhalis.17-28. (canceled)
 29. A method of producing a non-hemagglutinatingcultivar of M. catarrhalis from a HA M. catarrhalis strain or cultivar,which comprises serially passaging a HA M. catarrhalis strain orcultivar in static liquid cultures.
 30. The isolated antibody of claim14, which is a cytotoxic antibody that mediates complement killing ofMoraxella catarrhalis.